The RNA Interaction via secondary structure mediation (RNAmediator) tool suite analyses the change of RNA secondary structure upon binding other molecules.
It is available as suite of commandline tools, the source code of RNAmediator is open source and available via GitHub (License GPL-3).
RNAmediator can be installed with all tool dependencies via conda. Once you have conda installed simply type:
conda create -n RNAmediator -c conda-forge -c bioconda rnamediator
Activate the environment in which RNAmediator was installed to use it:
conda activate RNAmediator
RNAmediator provides different tools to answer different research questions. We will discuss the target application of the tools and the required input in the following points.
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ConstraintFold.py
Computes RNA secondary structure prediction with defined hard binding constraint for a set of sequences.
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Input:
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Output:
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ConstraintPLFold.py
Computes position-wise probability of being base-paired, using windows, with hard binding constraint for a set of sequences.
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Input:
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Output:
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CollectConsResults.py
Computes statistics for change of position-wise probability of being base-paired, with binding constraint for a set of provided genes.
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Input:
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Output:
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CollectWindowResults.py
Computes statistics for change of position-wise probability of being base-paired, using windows, with binding constraint for a set of provided genes.
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Input:
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Output:
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risvis.py
Visualize base pairing probabilties before and after constraining.
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Input:
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Output:
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ConstraintPLFold.py can be used for folding a list of sequences with constraints. Therefore it is recommended to use one fasta file containing sequences and another bed file containing the constraints. Matching between sequences and constraints is done via the gene identifier (here ENSG00000270742) which has to be the same in the bed file and the fasta and should look as follows:
>ENSG00000270742:chr1:61124732-61125202(+)
TTTTTTCTTTATAATTATTCCCCTATTTGAAAAATCAACTTGTATATGAGGCAGCAAACACCTTGCAGAGC...
chr1 26 35 ENSG00000270742 . +
The command line call for this simple scenario is supposed to look like this:
python ConstraintPLFold.py -s FASTA -x BedFileby default the results are printed to stdout. The first output is the
folded sequence without constraints. This output can be redirected to a file
using --unconstraint nameforunconstraint. The following outputs are the matrices for
an unpaired constraint and for an paired constraint. These matrices can also be
redirected to an file using --paired nameforpaired or --unpaired nameforunpaired.
The folder to which these files should be saved is determined via the --outdir flag.
Constraints can also be passed using the ono (one on one) flag like this:
python ConstraintPLFold.py -s FASTA -x ono,BedFileMatching between constraints and sequences consequently ignores the gene identifiers and instead the first sequence will use the first line in the bed file as a constraint.
If you want to construct bed files with genomic coordinates, containing information about
pairing probabilities (see CollectConsResults), it is essential to
provide another bed file with genomic coordinates.
This file can be passed using the --genes GeneBedFile and should look like:
chr1 61124732 61125202 ENSG00000270742 . +
If you plan to use transcripts, missing exons, instead of genes it is highly recommended changing the header of your fasta sequences as well as the constraints in the bed file as follows:
>ENST00000240304.5::ENST00000240304.5:0-5482(.)
GUCUUGUCGGCUCCUGUGUGUAGGAGGGAUUUCGGCCUGAGAGCGGGCCGAGGAGAUUGGCGACGGUGUCGCCCGUGUUUUCGUUGGCGGGUGCCUGGGCUGGUGGGAACAGCCGCCCGAAGGAAGCACCAUGAUUUCGGCCGCGCAGUUGUUGGAUGAGUUAAUGGGCCGGGACCGAAACCUAGCCCCGGACGAGAAGCGCAGCAACGUGCGGUGGGACCACGAGAGCGUUUGUAAAUAUUAUCUCUGUGGUUUUUGUCCUGCGGAAUUGUUCACAAAUACACGUUCUGAUCUUGGUCCGUGUGAAAAAAUUCAUGAUGAAAAUCUACGAAAACAGUAUGAGAAGAGCUCUCGUUUCAUGAAAGUUGGCUAUGAGAGAGAUUUUUUGCGAUACUUACAGAGCUUACUUGCAGAAGUAGAACGUAGGAUCAGACGAGGCCAUGCUCGUUUGGCAUUAUCUCAAAACCAGCAGUCUUCUGGGGCCGCUGGCCCAACAGGCAAAAAUGAAGAAAAAAUUCAGGUUCUAACAGACAAAAUUGAUGUACUUCUGCAACAGAUUGAAGAAUUAGGGUCUGAAGGAAAAGUAGAAGAAGCCCAGGGGAUGAUGAAAUUAGUUGAGCAAUUAAAAGAAGAGAGAGAACUGCUAAGGUCCACAACGUCGACAAUUGAAAGCUUUGCUGCACAAGAAAAACAAAUGGAAGUUUGUGAAGUAUGUGGAGCCUUUUUAAUAGUAGGAGAUGCCCAGUCCCGGGUAGAUGACCAUUUGAUGGGAAAACAACACAUGGGCUAUGCCAAAAUUAAAGCUACUGUAGAAGAAUUAAAAGAAAAGUUAAGGAAAAGAACCGAAGAACCUGAUCGUGAUGAGCGUCUAAAAAAGGAGAAGCAAGAAAGAGAAGAAAGAGAAAAAGAACGGGAGAGAGAAAGGGAAGAAAGAGAAAGGAAAAGACGAAGGGAAGAGGAAGAAAGAGAAAAAGAAAGGGCUCGUGACAGAGAAAGAAGAAAGAGAAGUCGUUCACGAAGUAGACACUCAAGCCGAACAUCAGACAGAAGAUGCAGCAGGUCUCGGGACCACAAAAGGUCACGAAGUAGAGAAAGAAGGCGGAGCAGAAGUAGAGAUCGACGAAGAAGCAGAAGCCAUGAUCGAUCAGAAAGAAAACACAGAUCUCGAAGUCGGGAUCGAAGAAGAUCAAAAAGCCGGGAUCGAAAGUCAUAUAAGCACAGGAGCAAAAGUCGGGACAGAGAACAAGAUAGAAAAUCCAAGGAGAAAGAAAAGAGGGGAUCUGAUGAUAAAAAAAGUAGUGUGAAGUCCGGUAGUCGAGAAAAGCAGAGUGAAGACACAAACACUGAAUCGAAGGAAAGUGAUACUAAGAAUGAGGUCAAUGGGACCAGUGAAGACAUUAAAUCUGAAGGUGACACUCAGUCCAAUUAAAACUGAUCUGAUAAGACCUCAGAUCAGACAGAGGACUACUGUUCGAAGAUUUUUGGAAGAAUACUGAGAACGGCAUAAAGUGAAGAUCGACAUUUAAAAAAUGAGGUGAAAGAAAGCUAUAGUGGCAUAGAAAAAGUAUAAAGCUCAGUUAGUUUUUUUAUUAUUAUUAUUAUUAAAAGUUAAUUCAGGACUGAUGUGACCUACCAGAUUUCAGAACAUGUGUUAAUAGUAUAUAUGCCACUGAAAACUUAGGUCCUGUAUCAUACUUUUUUCUUUAAGACUUUUUAAGAAAUAUUACUUAAACAUGUGGCUUGCUCAGUGUUUAAUUGCAAGUUUUCAAUCUUGGACUUUGAAAACAGGAUUAAACGUUAGUAUUCGUGUGAAUCAGACUAAGUGGGAUUUCAUUUUUACAACUCUGCUCUACUUAGCCUUUGGAUUUAGAAGUAAAAAUAAAGUAUCUCUGACUUUCUGUUACAAAGUUGAUUGUCUCUGUCAUUGAAAAGUUUUAGUAUUAAUCUUUUUCUAAUAAAGUUAUUGACUCUGAACUAGUCCCCUGUUUUAAAUACAAGAGUUACACUAUUACUAGAGGUGUUGGUGUACAGUUUUAUCUGAUUUGUUCUGUUUAAGACUAAUUUUUAUAGACUUUCUAAUGUUUUAAAUAAUGGUGCUUCAAUUUUAGGUGGUUAUGAAUAAAUUUGAAUUUUGCUUUUAAUAGCAAAGAUGUGCAGUGAACUAGAAUAUAUUUUUACAUCCCUGAGAGAUUCAUUUAGUAGAAAAUUCCAAGUAUCCUGACAAGCACUCUUUAGCUGGCUAGCUAUGGGAUGAUGUAGAAAAGCAUUCAAGAGCUAGUUUUUGUUAAGUCCUGUAUCAAGAUUAACCCAGCUGUGUCAGUUUAUAAAUGUAUUUGUGUAUAGGGUGUGUAGUAUAUAUGGCAAGGGUUUUUUCCCCCCACUUAAGUGAUUAUUUUUGUGUCACAUCUAGGAAAACCGGCAGCAUGUUUCUAUCUAUAGCCAGCUUCUUCGACUGUAUAAAAGUAUUCUCUCCAGCUACGUAUAUACACACAUACAUAUAUAUCAUAGCAAUUCCUUGUGGUUUAUAACUUGCAAAUACUGCUAUCAGUUUAUAGGUAAAGAAACAGUGUGUUAAAUGACUUAUCCAGGGAGGGUCCUGUGGCUUCAUGUUUAUGGAGUUGCUAGGUCUCUGCCUCAUGGUCCAGUGCCUGUUAAGCCACUGUGUUCAUUCUAAUAGGCAUAAUGAAUUGUUAAAGAAUUUACUAAAAUCUCUUCCACCAAACUUUGAAAAAUAAUGAAGCCGCCCCCACUUUAGAGGCUCUGUAUGAAAAAAUGCUGUGGAGACAGAGCCCUCCUGGCUCCCUAGCUGAUCCUGGAGAUGCAGCAAUAGAUGAAUGGGUUAUCUCUGAAUUUGUAAGAGAUAAUUCACAUGAGGAUUAAGAUAAAAUGGGAAGUAAAAUCUAACAAACACAAAGAUAGCUCCCAGGCACUGCUUUGUGUAGUUUGACAGCAUUGUGGUUGUAGCAGCAAAGGACUUAAAGUGAUAGUUUUUAAACCAUAUUCUGUCCCUAAGUAAUAAAAAAUCUAGGAAGUUACUAAAAUACCAGAUUUGUUCUGCUCUGCCUCAUCUAGAAUCAACGUCUAACUAACUUAAAUGAAGUAUAAUAAAUGAGUUCAUAUGAAAAGGCUUCCUCUAUGGACACUUAGAUAUAUUGUAACUAUUGAAGUUACCUGGGAUGUGGGGGUGGUGGGAGGAGGACCUGCCUCCCCAGGACAUCUAUGACUAAGGCCUGGCUUUAGUUAUGGAGAGAGACGUAGAAGUUGAAUUUUACACCCAAAAUUGAUGUGACUGAAGAGGAACUGAUUGUUGCUAACCAGCUCACAAGAAUCCAGUAUUGAGACCAGUUCACUAGAAGAAACAAACAUUUCUGCCAUGCAGACCAAAAAGUUAUUAGUUGGUGAAUAUGUAUUUUCUCUUUGGAAGGUCUUUAAGGGGAGCAAACCAGUUUUAAUCAAUCAGAUUGCUUGGUAAGUUUGGAAUCUGCAAUCAGUUGGUCUUAAAAAAAAAAAAACUUUAUUUUGGAAAUUUAAAGACAUACACAAAAGAGGAACAAUAUAAUUAACCUCUGUUAACUCAUCACCAACAAGACUCAUGACCACUUUUAUACUUCAUGAGUGAUUGUAUUUGUAUCCACUGUUUUCUAUUAUUUUCGAGCAAGUCUCAGACACACCAUUUAAUCUGUAAAUAAUUCAGCAUGUAUCUCUAAAAGACAAAGACCUCUUAAAUAACAGUUCAUUAGUAUAAAACAAAUUGGGUAAACUUUUGUUGGUCAUCAAACUAUAUUAGCACUGGUCCAAUAGUUUAAUUUUCAUUGAGCCUUUCAAGAGGACCGACCAGUCUGCUGCUCAAGACAUCCUCUCCUCUGGAAUGUAGAGAUAAUACAUAUCAUGCUCCUUUUUGUUAAAACGUUUUUUUUUCCCCUUCAAACACAGUCCAUUCAUUUUUCAGUUUGGGUUGAAACAUCCUUUUCUUGAUCUUGAGCUUAUAAUAACCUAGUCAUAUUGCUCAGCUCAGAUAUUUUUACUCCCUCUCCUUAGGCAUUCUGGUUCCUUAAAUAUAGUUAGUGUCACAGAGGAUAAAUAACCAACCUUAUUUCUAAGGUCUGAGAACACUUGGACCACAUAUUGGUUGAGCUCAGCCACCUUCUGAUUAAAGUUUUCAGACUUGUAAGAACUGAAAAUUUUUAUGGUGGAAGUUCUCUGAGCCCUCAUCCAUUCUGUUUUUAAAAAUGCAUUGCAGAUGGGCUAUGUGAAUAUGUUUUUAAACAUCUGAUAUGUGCAUGAAACAAAAAACACUUGAAGUUAUUAUGUAUACAAUUCUGUGGGAUGGGACUUCAUGCAGGAUUGGUUUUCAAGUUUGAUUUCCUGAGGGAUUUUUUAGUUGUUUGUGAAAGAACCCCAGGUCUACUUUUGAAAUUUUGUAUUAUAAUUGUAAUGUUGCCCAUGGUUAAAAAAAAAAAGUGUUCAGUGAUCUAUGUCUCCUACUACUCCUAUUUCUCUGUUUUUCCUCUGCAGGAGCUUGCUGCUGUUAACAGUUAUUCUUCCAAGUUGUUUUCUUUGUGGGGAGAUGGGAGGUGGGAGGAAAUAUAAACAUAUAUGUAUAGAUCUUUCAAAAUAUAUGACGGUAUACCCGUAUGUUCUGAGUCUUGCUGUUUUUACCUGGUAAUAUUUAGAAACAUUUAUUUUGAGAUAAAGGAGAGCACUUUUAAGUUGAACCUGUAGUUUUAAAAAGUACAUUUCAAGUAAGCCAAAGCAGAGAAGUAAAUGUAUUUUUCAUUGUUGUAUCAGAAUUUUGAAUUUACUAUUUUAAAAAUUCAAGAGUUUUGUAGCUGAUCUAUUUCUUCCCCUCAGCCAUCCCAAAUAGGUCAUUUGUCAACAGAUUUAAGAAUGUUUAGAAACAACAACUUUGGGAAACGGGAAACAAUUUGGUAUAAGUGGGUGUGCCAUAACCUCUCUCGUAGCCAUUCAUUCCCGGAUACAUACCCUAGAGAAACUCUUACACAUGCGUACCAGGGGAUGGAUUUAAGCAUUUGUGUGUAAUAGGAAGAAAAGAAGAAAAAACCCGGGAAGAUCCCAAGUGUCCACCAACAGUGUGUUGGAUAAAUACUGUGGUAUAUUCCAACAGUGGAAUUCCACAGAAGUGAAACUGAACUGCAGCUGUGUAUGUGAACAUGGACAAAACUCAACAAUAGAAGGAUCAAAAAAAGCAAGUCACAGAAGAAUACAUCACUAUGGUUCCAUUUCAAUGAAAGUCAAAAACAGGCUGUCAAAUACAUGAUAAAAGGAAACGAUUAAGACAAAAUUUAAUGUUAGCCGUUUUGAUGGAGGGAGAGGUGAUCAUGAGGGCACAGGGGUCUUCAGAAGAACUGGUGAGGGUCUGUUUCUGAAGCCUGUGGGCAUUUCCUUUUUUAAUCUGUAUGUUUAUGUGCUUUUGUAUGUAUGAUAUUUCUUAAUAAAAUUUAAAAAGAAGAAUGGGAAAAAA
ENST00000240304.5 1178 1183 ENST00000240304.5 . .
In this case you should use GeneBed files (--genes) which look quite similar to
the Constraints File but have as start position 0 and end position the length of
the sequence
ENST00000240304.5 0 5482 ENST00000240304.5 . .
The methods mentioned in ConstraintPLFold example will produce output that can be processed by CollectConsResults. This will generate BED files storing the probability of being unpaired for overlapping spans of nucleotides around the constraint. Therefore simply call:
python CollectConsResults.py -d path/to/ConstraintPLFold/output -u 5 -g GeneBed --outdir path/to/outdir --unconstraint name
Hereby it is essential that --unconstraint matches the uconstraint name provided at the ConstraintPLFold call.
Further the -u parameter defines the span sizes that are used for the output BED files which might
for example look like this for -u 5:
chr1 110135760 110135765 ENSG00000065135|44542-44544|110135774-110135777 0.02620831 + 9 0.05007846 0.07628677 2.2444807817789587 0.02620831000000001 0.04615814895706037 0.15227569
chr1 110135761 110135766 ENSG00000065135|44542-44544|110135774-110135777 0.019514269999999993 + 8 0.049938 0.06945227 2.426255722126518 0.019514269999999993 -0.03666320494171017 0.15227569
chr1 110135762 110135767 ENSG00000065135|44542-44544|110135774-110135777 0.09578122 + 7 0.13804191 0.23382313 1.4457214540425338 0.09578122 0.9069421599395611 0.15227569
chr1 110135763 110135768 ENSG00000065135|44542-44544|110135774-110135777 0.07929997 + 6 0.06154513 0.1408451 1.5621026141257632 0.07929996999999998 0.7030295094408919 0.15227569
chr1 110135778 110135783 ENSG00000065135|44542-44544|110135774-110135777 0.31023007 + -7 0.14063798 0.45086805 0.7213795423617754 0.31023007 3.560189541047878 0.15227569
chr1 110135779 110135784 ENSG00000065135|44542-44544|110135774-110135777 0.2991335 + -8 0.1657577 0.4648912 0.7438289320089759 0.2991335 3.4228983161623856 0.15227569
chr1 110135780 110135785 ENSG00000065135|44542-44544|110135774-110135777 0.29541885 + -9 0.15833754 0.45375639 0.7515304743397738 0.29541885 3.376939173064934 0.15227569
A detailed description of that files columns can be found in the Output section.
The BED file created by CollectConsResults can be used to intersect with other known binding sites on the same gene/transcript. Thus, it is possible to see whether the changes in RNA structure upon binding of one ligand might affect the structure of binding site of other ligands.